Experimental Animal Science

Comparative analysis of the structure and chromoso- mal assignment of CDI: an evidence for different evolutionary histories between classic CD1 and CD1D class genes

AKIHIRO MATSUURA 1 , MIYUKI KINEBUCHI j , SHIGEO KATABAMI 1 ,

CHEN HONG-ZHI 1, KIYOSHI KASAI 1 , SHINGO ICHIMIYA 1 ,

KAZUHIKO YAMADA 2, MICHIHIRO C. YOSHIDA 2, MASATO HORIE 3, and

NORIYUKI SATO !

l Department of Pathology, School of Medicine, Sapporo Medical University, Sapporo, Japan

2 Chromosome Research Unit, Faculty of Science, Hakkaido University, Sapporo, Japan 30htsuka Genome Research Institute, Tokushima, Japan

Summary

The non-MHC-encoded CD1 family has recently emerged as a novel antigen-presenting system that is distinct from MHC class I and class II molecules. In the present study, we determined the genomic structure of that rat CD1, and compared with those of other previously reported CD1 genes. Rat CDI was extremely similar to mouse CD1 genes, especially to CD1D1. It is of interest that a tyrosine-based motif for endosomal localization, identified in the human CDlb cytoplasmic tail, was conserved in all CD 1 molecules except for CD 1 a, that was encoded by a single short exon. Comparison of the overall exon-intron organization of CD1 genes revealed that the length of the introns was also characteristic to each of the two classes of CD1 genes; classic (CDIA, CDIB, CD1C and CD1E), and CD1D, which have been categorized by comparison of coding regions. These findings support a hypothesis that the two classes have different evolutionary histories. In contrast to the absence of the classic CD1 genes in rats and mice, the entire region of nonpolymor- phic CD1D gene has been conserved through mammalian evolution. Furthermore, we determined chromosomal localization of rat CD1 gene using the fluorescence in situ hybridization method with several probes derived from genomic rat CD1 clones. Similar to human and mouse CD1, rat CD1 mapped outside the MHC loci despite the structural and functional resemblance to MHC. Con- served syntheny of chromosomal segments of RNO2 and MMU3 is implied.

Key words: CD1, gene organization, evolution

This work was supported in part by a fund of Hokkaido Geriatrics Research Institute.

J. Exp. Anim. Sci. 2000; 41:87-90 Urban & Fischer Verlag http://www.urbanfischer.de/journals/jeansc 0939-8600/00/41/01-02-087 $12.00/0

88 A. MATSUURA et al.

Results and discussion

The CD1 family genes have been found in many mammalian species such as mice, rats, rabbits and sheep (BRADBURY et al. 1988, CALABI et al. 1989a, CALABI et al. 1989b,

FERGUSON et al. 1996, ICHIMIYA et al. 1994). We determined genomic organization of rat CD1 locus (KATABAMI et al. 1998) and compared with those of previously published CD1

genes. Structural organizations of CD1 and H2-K were shown in Fig. 1. All CD1 gene

contained six exons; exon 1 for the 5' untranslated region (5 'UN) and the leader peptide;

exons 2-4 for the ~1-3 domains, respectively; exon 5 for the transmembrane region and part of the cytoplasmic tail; and exon 6 for the remainder of the short cytoplasmic tail and the 3' untranslated region (3'UN). These results indicated that the overall exon-intron

organization of the CD1 gene was similar to that of the MHC class I gene, in that individ-

ual functional domains were encoded by separate exons. Comparison of the introns pro- vides an interesting view as to the evolution of CD1 genes found in different species.

Overall organization, including intronic length, could be divided into two types, which

were correlated with the previous categorization of two classes of CD1 genes, classic CD1 (including hCD1A, hCDIB, hCD1C and hCD1E) and CD1D (including mCD1D1,

mCD1D2 and rCD1). For example, the length of intron 1, about 280 bp in CD1D, was

shorter than the about 350 bp in classic CD1. The length of intron 2 in CD1D, about 280

bp, was also shorter than the about 600 bp in the classic CD1. The lengths of intron 3 and intron 4 of CD1D class genes were longer than those of classic CD1 with only a few

ex 1 ex 2 ex 3 ex 4 ex 5

intron 3

ex 6

CD1 D class

ex 1 ex 2 ex 3 ex 4 ex 5 ex 6

intron 3

classic CD1

ex 1 ex 2 ex 3 B2-Alu ex 4 5 6 7 ex 8

- E ] - t - - ~ - - I ~ r - -143-Gf134- -_~- - in t ron 3

H2-K

Fig. 1. Genomic organization of CD1D, classic CD1 and H2-K. Exons and introns are indicated by boxes and straigth lines, respectively. Comparison of CD1 gene categorized them into two classes based on composition of exonic sequences as well as length of introns. The CD1D class genes include rat CD1, mouse CD1D1 and CDID2, and human CD1D. The classic CD1 class genes include human CDIA, CDIB, CDIC, and CDIE. Organization of H2- K is shown as a representative of the classical MHC class I gene.

CD1 gene organization and evolution 89

exceptions; mCD1D2 had a deletion in intron 3 and hCDIA had an Alu repeat in intron 3.

Similarly, other intron length were also typical of the two classes.

The cytoplasmic portion of the CD1 molecule was encoded by the 3' end of the fifth

exon and short sixth exon; a stretch of charged amino acid residues, RRR in rat CD1, was followed by consensus sequence YQXI/V, YQDI in rat CD1. These features were well conserved in all CD1 molecules except for CDla. This tyrosine-based motif, YQNI, was

recently reported to be a signal for internalization and targeting of human CDlb antigens

to endosomal compartments (MIICs, MHC class II compartments) where antigen-loading

of class II molecules by endocytosed peptide occurs (SuGrrA et al. 1996). CDIa

molecules lost this motif by a point mutation at the eodon for Q (CAA) to a stop codon

(TAA) in the sixth exon, indicating that prototypical CD1 might carry this motif and be used for trafficking tO the endocytic system.

Based on these observation a model of CD1 evolution was depicted in Fig. 2. Ances-

tral CD1 gene might be CD1D-like and contain endosomal localization signal (shown by asterisk). Gene duplication resulted in generation of two prototypical CD1 classes. The

CD1D has been conserved through mammalian evolution and is implicated in a role in

innate immunity as CDld molecules act as the ligand for NKT cells with an invariant T

duplicalion l

"CDld-like"

delele classic C D 1 / in rodenl radiatione

/ ~ duplicalion

rCdld mCdldl mCdld2

Rat (RNO2) Mouse (MMU3)

ancestral CDt

"cIassic CDl-like"

\

mulliplication

hCdld hCdla hC~lc hCdlb hCdle

Human (HSA1)

~ liplicaticm

----5

J (

Rabbit Bovine Sheep

Fig. 2. A model of CD1 family gene evolution in mammals. Open and shaded boxes indicate CDlD-like and classic CDl-like genes, respectively. Asterisk indi- cates an endosomal localization signal near the carboxyl terminal of the cytoplasmic domain which was encoded by a single short exon 6. CD1A does not contain this signal. All species harbor single or only few copies of CDID. Rodents lack classic CD1, but most species have multiple copies of classic CD1.

90 A. MATSUURA et al.

cell receptor. The other classic CD1 has been deleted in rodents but expanded in other

species and is implicated in infectious immunity especially for protection against

mycobacterium. In humans, three classic CD1 showed tissue-specific expression by thy-

mus and antigen-presenting cells. Such a characteristic feature has also been observed for

classic CD1 in different species. It might be due to acquisition of the 5' regulatory ele-

ments quite early in CD1 evolution. In contrast, exclusion of CDla from endocytic path-

ways by truncation of cytoplasmic portion arouse recently perhaps in the primate radia-

tion.

By using fluorescence in situ hybridization, we have localized the rat CD1 to chromo-

some 2@4 (MATSUURA et al. 1999). A conserved gene linkage has been identified

between humans and mice. A chromosomal segment of HSA1 was splitted into MMU1

and MMU3. CD1 localized near this border. Similar to the mouse, a large linkage group

of HSA1 was splitted into RNO13 and RNO2.

References

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CALABI, F., J. M. JARVIS, L. MARTIN, and C. MILSTEIN. 1989b. Two classes of CD1 genes. Eur J Immunol 19: 285-292.

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ICHIMIYA, S., K. KXKUCHI, and A. MATSUURA. 1994. Structural analysis of the rat homologue of CDI: Evidence for evolutionary conservation of the CD1D class and widespread transcription by rat cells. J Immunol. 153:1112-1123.

KATABAMI, S., A. MATSUURA, H.-Z. CHEN, K. IMAI, and K. KIKUCHI. 1998. Structural organization of rat CD 1 typifies evolutionarily conserved CD 1D class genes. Immunogenetics 48:22-31.

MATSUURA, A., M. KINEBUCHI, S. KATABAMI, H. CHEN, K. YAMADA, M. C. YOSUIDA, M. HORIE, K. T. WATANABE, K. KIKUCHI, and N. SATO. 1999. Correction and confirmation of the assignment of the evolutionarily conserved CD1D class gene to rat chromosome 2q34 and its relationship to human and mouse loci. Cytogenet. Cell Genet, in press.

SUGITA, M., R. M. JACKMAN, E. VAN DONSELAAR, S. M. BEHAR, R. A. ROGERS, P. J. PETERS~ M. B. BRENNER, and S. A. PORCELLI. 1996. Cytoplasmic tail-dependent localization of CDlb antigen- presenting molecules to MIICs. Science 273: 349-352.

Corresponding author: Dr. AKIHIRO MATSUURA, Department of Pathology, School of Medicine, Sapporo Medical University, South-l, West-17, Chuo-ku, Sapporo 060-8856, Japan Phone: +81-11-611-2111 extension 2691; Fax: +81-11-643-2310; e-mail: amatsuur@ sapmed.ac.jp